Stabilized microbubble compositions

ABSTRACT

A microbubble preparation formed of a plurality of microbubbles comprising a first gas and a second gas surrounded by a membrane such as a surfactant, wherein the first gas and the second gas are present in a molar ratio of from about 1:100 to about 1000:1, and wherein the first gas has a vapor pressure of at least about (760−x) mm Hg at 37° C., where x is the vapor pressure of the second gas at 37° C., and wherein the vapor pressure of each of the first and second gases is greater than about 75 mm Hg at 37° C.; also disclosed are methods for preparing microbubble compositions, including compositions that rapidly shrink from a first average diameter to a second average diameter less than about 75% of the first average diameter and are stabilized at the second average diameter; kits for preparing microbubbles; and methods for using such microbubbles as ultrasound contrast agents

RELATED APPLICATIONS

[0001] This application is a continuation of U.S. Ser. No. 08/785,007filed Jan. 17, 1997, which is a continuation of U.S. Ser. No.08/405,447, filed Mar. 16, 1995, now U.S. Pat. No. 5,639,443, which is acontinuation of U.S. Ser. No. 08/099,951, filed Jul. 30, 1993 nowabandoned.

FIELD OF THE INVENTION

[0002] The present invention includes a method for preparing stablelong-lived microbubbles for ultrasound contrast enhancement and otheruses, and to compositions of the bubbles so prepared.

BACKGROUND OF THE ART

[0003] Ultrasound technology provides an important and more economicalalternative to imaging techniques which use ionizing radiation. Whilenumerous conventional imaging technologies are available, e.g., magneticresonance imaging (MRI), computerized tomography (CT), and positronemission tomography (PET), each of these techniques use extremelyexpensive equipment. Moreover, CT and PET utilize ionizing radiation.Unlike these techniques, ultrasound imaging equipment is relativelyinexpensive. Moreover, ultrasound imaging does not use ionizingradiation.

[0004] Ultrasound imaging makes use of differences in tissue density andcomposition that affect the reflection of sound waves by those tissues.Images are especially sharp where there are distinct variations intissue density or compressibility, such as at tissue interfaces.Interfaces between solid tissues, the skeletal system, and variousorgans and/or tumors are readily imaged with ultrasound.

[0005] Accordingly, in many imaging applications ultrasound performssuitably without use of contrast enhancement agents; however, for otherapplications, such as visualization of flowing blood in tissues, therehave been ongoing efforts to develop such agents to provide contrastenhancement. One particularly significant application for such contrastagents is in the area of vascular imaging. Such ultrasound contrastagents could improve imaging of flowing blood in the heart, kidneys,lungs, and other tissues. This, in turn, would facilitate research,diagnosis, surgery, and therapy related to the imaged tissues. A bloodpool contrast agent would also allow imaging on the basis of bloodcontent (e.g., tumors and inflamed tissues) and would aid in thevisualization of the placenta and fetus by enhancing only the maternalcirculation.

[0006] A variety of ultrasound contrast enhancement agents have beenproposed. The most successful agents have generally consisted ofmicrobubbles that can be injected intravenously. In their simplestembodiment, microbubbles are miniature bubbles containing a gas, such asair, and are formed through the use of foaming agents, surfactants, orencapsulating agents. The microbubbles then provide a physical object inthe flowing blood that is of a different density and a much highercompressibility than the surrounding fluid tissue and blood. As aresult, these microbubbles can easily be imaged with ultrasound.

[0007] Most microbubble compositions have failed, however, to providecontrast enhancement that lasts even a few seconds, let alone minutes,of contrast enhancement. This greatly limits their usefulness.Microbubbles have therefore been “constructed” in various manners in anattempt to increase their effective contrast enhancement life. Variousavenues have been pursued: use of different surfactants or foamingagents; use of gelatins or albumin microspheres that are initiallyformed in liquid suspension, and which entrap gas during solidification;and liposome formation. Each of these attempts, in theory, should act tocreate stronger bubble structures. However, the entrapped gases(typically air, CO₂, and the like) are under increased pressure in thebubble due to the surface tension of the surrounding surfactant, asdescribed by the LaPlace equation (ΔP=2γ/r).

[0008] This increased pressure, in turn, results in rapid shrinkage anddisappearance of the bubble as the gas moves from a high pressure area(in the bubble) to a lower pressure environment (in either thesurrounding liquid which is not saturated with gas at this elevatedpressure, or into a larger diameter, lower pressure bubble).

[0009] Solid phase shells that encapsulate gases have generally proventoo fragile or to permeable to the gas to have satisfactory in vivolife. Furthermore, thick shells (e.g., albumin, sugar, or other viscousmaterials) reduce the compressibility of the bubbles, thereby reducingtheir echogenicity during the short time they can exist. Solid particlesor liquid emulsion droplets that evolve gas or boil when injected posethe danger of supersaturating the blood with the gas or vapor. This willlead to a small number of large embolizing bubbles forming at the fewavailable nucleation sites rather than the intended large number ofsmall bubbles.

[0010] One proposal for dealing with such problems is outlined in Quay,PCT/US92/07250. Quay forms bubbles using gases selected on the basis ofbeing a gas at body temperature (below 37° C.), and having reduced watersolubility, higher density, and reduced gas diffusivity in solution incomparison to air. Although reduced water solubility and diffusivity canaffect the rate at which the gas leaves the bubble, numerous problemsremain with the Quay bubbles. Forming bubbles of sufficiently smalldiameter (e.g., 0.2 μm) requires high energy input. This is adisadvantage in that sophisticated bubble preparation systems must beprovided at the site of use. Moreover, The Quay gas selection criteriaare incorrect in that they fail to consider certain major causes ofbubble shrinkage, namely, the effects of bubble surface tension,surfactants and gas osmotic effects, and these errors result in theinclusion of certain unsuitable gases and the exclusion of certainoptimally suitable gases.

[0011] Accordingly, a need exists in the art for compositions, and amethod to prepare such compositions, that provide, or utilize, a longerlife contrast enhancement agent that is biocompatible, easily prepared,and provides superior contrast enhancement in ultrasound imaging.

SUMMARY OF THE INVENTION

[0012] In accordance with the present invention, there is provided anultrasound contrast enhancement agent that has a prolonged longevity invivo, which consists of virtually any conventional microbubbleformulation in conjunction with an entrapped gas or gas mixture that isselected based upon consideration of partial pressures of gases bothinside and outside of the bubble, and on the resulting differences ingas osmotic pressure that oppose bubble shrinkage. Gases having a lowvapor pressure and limited solubility in blood or serum (i.e.,relatively hydrophobic) may advantageously be provided in combinationwith another gas that is more rapidly exchanged with gases present innormal blood or serum. Surfactant families allowing the use of highermolecular weight gas osmotic agents, and improved methods of bubbleproduction are also disclosed.

[0013] One aspect of the present invention is a stabilized gas filledmicrobubble preparation, comprising a mixture of a first gas or gasesand a second gas or gases within generally spherical membranes to formmicrobubbles, wherein the first gas and the second gas are respectivelypresent in a molar ratio of about 1:100 to about 1000:1, and wherein thefirst gas has a vapor pressure of at least about (760−x) mm Hg at 37°C., where x is the vapor pressure of the second gas at 37° C., andwherein the vapor pressure of each of the first and second gases isgreater than about 75 mm Hg at 37° C., with the proviso that the firstgas and the second gas are not water vapor. In one embodiment, thesecond gas comprises a fluorocarbon and the first gas is anonfluorocarbon, such as nitrogen, oxygen, carbon dioxide, or a mixturethereof.

[0014] The microbubbles may advantageously be provided in a liquidmedium, such as an aqueous medium, wherein they have a first averagediameter, the ratio of the first gas to the second gas in themicrobubbles is at least 1:1, and the microbubbles are adapted to shrinkin the medium as a result of loss of the first gas through the membraneto a second average diameter of less than about 75% of the firstdiameter and then remain stabilized at or about the second diameter forat least about 1 minute as a result of a gas osmotic pressuredifferential across the membrane. Advantageously, the medium is in acontainer and the microbubbles have actually been formed in thecontainer. Alternatively, the medium is blood in vivo. In oneembodiment, the liquid medium contains gas or gases dissolved thereinwith a gas tension of at least about 700 mm Hg, wherein the firstdiameter is at least about 5 μm, and wherein the tension of the gas orgases dissolved in the medium is less than the partial pressure of thesame gas or gases inside the microbubbles.

[0015] In a particularly preferred embodiment, the bubble initiallycontains at least three gases: a first gas having a partial pressure fargreater than the gas tension of the same gas in the surrounding liquid(e.g., 1.5, 2, 4, 5, 10, 20, 50, or 100 or more times greater than inthe surrounding liquid); a second gas that is retained in the bubble dueto a relatively low permeability of the bubble membrane to the gas, or arelatively low solubility of the gas in the surrounding medium (asdescribed elsewhere herein), and a third gas to which the membrane isrelatively permeable that is also present in the surrounding medium. Forexample, in an aqueous system exposed to or at least partiallyequilibrated with air (such as blood), the first gas may advantageouslybe carbon dioxide or another gas not present in large quantities in airor blood; the second gas may be a fluorocarbon gas, such asperfluorohexane; and the third gas may be air or a major component ofair such as nitrogen or oxygen.

[0016] Preferably, the first diameter prior to shrinkage is at leastabout 10 μm and the second diameter at which the diameter is stabilizedis between about 1 μm and 6 μm.

[0017] For all of the microbubble preparations or methods describedherein, in one preferred embodiment, the second gas has an averagemolecular weight at least about 4 times that of the first gas. Inanother preferred embodiment, the second gas has a vapor pressure lessthan about 750 or 760 mm Hg at 37° C. Moreover, it is preferred that themolar ratio of the first gas to the second gas is from about 1:10 toabout 500:1, 200:1, or 100:1. In other preferred embodiments, the secondgas comprises a fluorocarbon or a mixture of at least two or threefluorocarbons, and the first gas is a nonfluorocarbon. In someadvantageous preparations, the second gas comprises one or moreperfluorocarbons. In others, both the first gas and the second gascomprise fluorocarbons. In still others, the microbubbles contain as thefirst gas, or as the second gas, or respectively as the first and secondgases, gaseous perfluorobutane and perfluorohexane in a ratio from about1:10 to about 10:1. Alternatively, the microbubbles contain as the firstgas, or as the second gas, or respectively as the first and secondgases, gaseous perfluorobutane and perfluoropentane in a ratio fromabout 1:10 to about 10:1. It is advantageous that the second gas leavethe microbubble much more slowly than does the first gas; thus, it ispreferred that the second gas has a water solubility of not more thanabout 0.5 mM at 25° C. and one atmosphere, and the first gas has a watersolubility at least about 10 times, and preferably at least 20, 50, 100,or 200 times greater than that of the second gas. Similarly, it ispreferred that the permeability of the membrane to the first gas is atleast about 5 times, preferably 10, 20, 50, or 100 times greater thanthe permeability of the membrane to the second gas.

[0018] The microbubble preparation may advantageously be contained in acontainer, having a liquid in the container in admixture with themicrobubbles, wherein the container further comprises means fortransmission of sufficient ultrasonic energy to the liquid to permitformation of the microbubbles by sonication. In this way, themicrobubbles can be formed by the physician (or other professional)immediately before use by applying ultrasonic energy from an outsidesource to the sterile preparation inside the container. This means fortransmission can, for example, be a flexible polymer material having athickness less than about 0.5 mm (which permits ready transmission ofultrasonic energy without overheating the membrane). Such membranes canbe prepared from such polymers as natural or synthetic rubber or otherelastomer, polytetrafluoroethylene, polyethylene terephthalate, and thelike.

[0019] In the microbubble preparations of the invention, the membraneenclosing the gas is preferably a surfactant. One preferred type ofsurfactant comprises a non-Newtonian viscoelastic surfactant, alone orin combination with another surfactant. Other preferred general andspecific categories of surfactants include carbohydrates, such aspolysaccharides, derivatized carbohydrates, such as fatty acid esters ofsugars such as sucrose (preferably sucrose stearate), and proteinaceoussurfactants including albumin. Alternatively, the membrane of themicrobubble need not be a fluid (such as a surfactant), but instead canbe a solid or semi-solid, such as hardened, thickened, or denaturedproteinaceous material (e.g. albumin), carbohydrates, and the like.

[0020] One advantageous form of the invention is a kit for use inpreparing microbubbles, preferably at the site of use. This kit maycomprise sealed container (such as a vial with a septum seal for easyremoval of the microbubbles using a hypodermic syringe), a liquid in thecontainer (such as water or a buffered, isotonic, sterile aqueousmedium), a surfactant in the container, and a fluorocarbon gas(including a fluorocarbon vapor) in the container, wherein the liquid,the surfactant, and the fluorocarbon gas or vapor are together adaptedto form microbubbles upon the application of energy thereto. The energyadvantageously may be simple shaking energy, either manual ormechanical, stirring or whipping, or ultrasonic energy. The kitpreferably includes means in the container for permitting transmissionof sufficient external ultrasonic energy to the liquid to formmicrobubbles in the container. As above, the means for transmission canin one embodiment comprise a flexible polymer membrane having athickness less than about 0.5 mm. In one embodiment, the kit furtherincludes a nonfluorocarbon gas in the container, wherein the molar ratioof the nonfluorocarbon gas to the fluorocarbon gas is from about 1:10 toabout 1000:1, with the proviso that the nonfluorocarbon gas is not watervapor. In all of the kits of the present invention, the surfactant, thegas or gases, and the other elements of the kit may in some embodimentsbe the same as recited above for the microbubble preparation per se.

[0021] In another embodiment, the kit comprises a container, driedliquid-soluble void-containing structures in the container, thevoid-containing structures defining a plurality of voids having anaverage diameter less than about 100 μm, a gas in the voids, and asurfactant, wherein the void-containing structures, the gas, and thesurfactant are together adapted to form microbubbles upon addition tothe container of a liquid in which the void-containing structures aresoluble. These void-containing structures can be made at least in partof the surfactant, e.g., by lyophilization of void-forming material orby spray drying, or can be formed from any other liquid soluble(preferably water soluble) film-forming material, such as albumin,enzymes, or other proteins, simple or complex carbohydrates orpolysaccharides, and the like. The surfactants used in the kit canadvantageously be those described above in connection with themicrobubble preparations per se.

[0022] The present invention also includes a method for formingmicrobubbles, comprising the steps of providing a first gas, a secondgas, a membrane forming material, and a liquid, wherein the first gasand the second gas are present in a molar ratio of from about 1:100 toabout 1,000:1, and wherein the first gas has a vapor pressure of atleast about (760−x) mm Hg at 37° C., where x is the vapor pressure ofthe second gas at 37° C., and wherein the vapor pressure of each of thefirst and second gases is greater than about 75 mm Hg at 37° C., withthe proviso that the first gas and the second gas are not water vapor,and surrounding the first and second gases with the membrane formingmaterial to form microbubbles in the liquid. The membrane formingmaterials and gases may be as described above. The method preferablyfurther comprises the steps of initially forming microbubbles having afirst average diameter wherein the initial ratio of the first gas to thesecond gas in the microbubbles is at least about 1:1, contacting themicrobubbles having a first average diameter with a liquid medium,shrinking the microbubbles in the medium as a result of loss of thefirst gas through the membrane, and then stabilizing the microbubbles ata second average diameter of less than about 75% of the first diameterfor a period of at least one minute. Preferably, the microbubbles arestabilized at the second diameter by providing a gas osmotic pressuredifferential across the membrane such that the tension of a gas or gasesdissolved in the medium is greater than or equal to the pressure of thesame gas or gases inside the microbubbles. In one embodiment, the firstdiameter is at least about 5 μm.

[0023] The invention also includes a method for forming microbubbles,comprising the steps of providing dried liquid-soluble void-containingstructures, the void-containing structures defining a plurality of voidshaving a diameter less than about 100 μm, providing a gas in the voids,providing a surfactant, combining together the void-containingstructures, the gas, the surfactant, and a liquid in which thevoid-containing structures are soluble, and dissolving thevoid-containing structures in the liquid whereby the gas in theenclosures forms microbubbles that are surrounded by the surfactant. Aswith the kit, preferred void-containing structures are formed ofprotein, surfactant, carbohydrate, or any of the other materialsdescribed above.

[0024] Finally, the present invention includes a method for imaging anobject or body, comprising the steps of introducing into the object orbody any of the aforementioned microbubble preparations and thenultrasonically imaging at least a portion of the object or body.Preferably, the body is a vertebrate and the preparation is introducedinto the vasculature of the vertebrate. The method may further includepreparing the microbubbles in any of the aforementioned manners prior tointroduction into the animal.

DETAILED DESCRIPTION OF THE INVENTION

[0025] As used in the present description and claims, the terms “vapor”and “gas” are used interchangeably. Similarly, when referring to thetension of dissolved gas in a liquid, the more familiar term “pressure”may be used interchangeably with “tension.” “Gas osmotic pressure” ismore fully defined below, but in a simple approximation can be thoughtof as the difference between the partial pressure of a gas inside amicrobubble and the pressure or tension of that gas (either in a gasphase or dissolved in a liquid phase) outside of the microbubble, whenthe microbubble membrane is permeable to the gas. More precisely, itrelates to differences in gas diffusion rates across a membrane. Theterm “membrane” is used to refer to the material surrounding or defininga microbubble, whether it be a surfactant, another film forming liquid,or a film forming solid or semisolid. “Microbubbles” are considered tobe bubbles having a diameter between about 0.5 and 300 μm, preferablyhaving a diameter no more than about 200, 100, or 50 μm, and forintravascular use, preferably not more than about 10, 8, 7, 6, or 5 μm(measured as average number weighted diameter of the microbubblecomposition). When referring to a “gas,” it will be understood thatmixtures of gases together having the requisite property fall within thedefinition, except where the context otherwise requires. Thus, air maytypically be considered a “gas” herein.

[0026] The present invention provides microbubbles that have a prolongedlongevity in vivo that are suitable for use as ultrasound contrastenhancement agents. Typical ultrasound contrast enhancement agentsexhibit contrast enhancement potential for only about one pass throughthe arterial system, or a few seconds to about a minute, and thus do notsurvive past the aorta in a patient following intravenous injection. Incomparison, contrast agents prepared in accordance with the presentinvention continue to demonstrate contrast enhancements lives measuredin multiple passes through the entire circulatory system of a patientfollowing intravenous injection. Bubble lives of several minutes areeasily demonstrated. Such lengthening of contrast enhancement potentialduring ultrasound is highly advantageous. In addition, the contrastenhancement agents of the invention provide superior imaging; forexample, clear, vivid, and distinct images of blood flowing through theheart, lungs, and kidneys are achieved. Thus small, nontoxic doses canbe administered in a peripheral vein and used to enhance images of theentire body.

[0027] While bubbles have been shown to be the most efficient ultrasoundscatterers for use in intravenous ultrasound contrast agents, their mainpractical drawback is the extremely short lifetime of the small(typically less than 5 microns diameter) bubbles required to passthrough capillaries in suspension. This short lifetime is caused by theincreased gas pressure inside the bubble, which results from the surfacetension forces acting on the bubble. This elevated internal pressureincreases as the diameter of the bubble is reduced. The increasedinternal gas pressure forces the gas inside the bubble to dissolve,resulting in bubble collapse as the gas is forced into solution. TheLaPlace equation, ΔP=2γ/r, (where ΔP is the increased gas pressureinside the bubble, γ is the surface tension of the bubble film, and r isthe radius of the bubble) describes the pressure exerted on a gas bubbleby the surrounding bubble surface or film. The LaPlace pressure isinversely proportional to the bubble radius; thus, as the bubbleshrinks, the LaPlace pressure increases, increasing the rate ofdiffusion of gas out of the bubble and the rate of bubble shrinkage.

[0028] It was surprisingly discovered that gases and gas vapor mixtureswhich can exert a gas osmotic pressure opposing the LaPlace pressure cangreatly retard the collapse of these small diameter bubbles. In general,the invention uses a primary modifier gas or mixture of gases thatdilute a gas osmotic agent to a partial pressure less than the gasosmotic agent's vapor pressure until the modifier gas will exchange withgases normally present in the external medium. The gas osmotic agent oragents are generally relatively hydrophobic and relatively bubblemembrane impermeable and also further possess the ability to develop gasosmotic pressures greater than 75 or 100 Torr at a relatively low vaporpressure.

[0029] The process of the invention is related to the well known osmoticeffect observed in a dialysis bag containing a solute that issubstantially membrane impermeable (e.g. PEG, albumin, polysaccharide,starch) dissolved in an aqueous solution is exposed to a pure waterexternal phase. The solute inside the bag dilutes the water inside thebag and thus reduces the rate of water diffusion out of the bag relativeto the rate of pure water (full concentration) diffusion into the bag.The bag will expand in volume until an equilibrium is established withan elevated internal pressure within the bag which increases the outwarddiffusional flux rate of water to balance the inward flux rate of thepure water. This pressure difference is the osmotic pressure between thesolutions.

[0030] In the above system, the internal pressure will slowly drop asthe solute slowly diffuses out of the bag, thus reducing the internalsolute concentration. Other materials dissolved in the solutionsurrounding the bag will reduce this pressure further, and, if they aremore effective or at a higher concentration, will shrink the bag.

[0031] It was observed that bubbles of air saturated with selectedperfluorocarbons grow rather than shrink when exposed to air dissolvedin a liquid due to the gas osmotic pressure exerted by theperfluorocarbon vapor. The perfluorocarbon vapor is relativelyimpermeable to the bubble film and thus remains inside the bubble. Theair inside the bubble is diluted by the perfluorocarbon, which acts toslow the air diffusion flux out of the bubble. This gas osmotic pressureis proportional to the concentration gradient of the perfluorocarbonvapor across the bubble film, the concentration of air surrounding thebubble, and the ratio of the bubble film permeability to air and toperfluorocarbon.

[0032] As discussed above, the LaPlace pressure is inverselyproportional to the bubble radius; thus, as the bubble shrinks, theLaPlace pressure increases, increasing the rate of diffusion of gas outof the bubble and the rate of bubble shrinkage, and in some casesleading to the condensation and virtual disappearance of a gas in thebubble as the combined LaPlace and external pressures concentrate theosmotic agent until its partial pressure reaches the vapor pressure ofliquid osmotic agent.

[0033] We have discovered that conventional microbubbles that containany single gas will subsist in the blood for a length of time thatdepends primarily on the arterial pressure, the bubble diameter, themembrane permeability of the gas through the bubble's surface, themechanical strength of the bubble's surface, the presence, absence, andconcentration of the gases that are ordinarily present in the blood orserum, and the surface tension present at the surface of the bubble(which is primarily dependent on the diameter of the bubble andsecondarily dependent on the identity and concentration of thesurfactants which form the bubble's surface). Each of these parametersare interrelated, and they interact in the bubble to determine thelength of time that the bubble will last in the blood.

[0034] The present invention includes the discovery that a single gas ora combination of gases can together act to stabilize the structure ofthe microbubbles entraining or entrapping them. Essentially, theinvention utilizes a first gas or gases (a “primary modifier gas”) thatoptionally is ordinarily present in normal blood and serum incombination with one or more additional second gases (a “gas osmoticagent or agents” or a “secondary gas”) that act to regulate the osmoticpressure within the bubble. Through regulating the osmotic pressure ofthe bubble, the gas osmotic agent (defined herein as a single or mixtureof chemical entities) exerts pressure within the bubble, aiding inpreventing deflation. Optionally, the modifier gas may be a gas that isnot ordinarily present in blood or serum. However, the modifier gas mustbe capable of diluting and maintaining the gas osmotic agent or agentsat a partial pressure below the vapor pressure of the gas osmotic agentor agents while the gases in blood or other surrounding liquid diffuseinto the bubble. In an aqueous medium, water vapor is not considered tobe one of the “gases” in question. Similarly, when microbubbles are in anonaqueous liquid medium, the vapor of that medium is not considered tobe one of the “gases.”

[0035] We have discovered that by adding a gas osmotic agent that has,for example, a reduced membrane permeability through the bubble'ssurface or reduced solubility in the external continuous phase liquidphase, the life of a bubble formed therewith will be radicallyincreased. This stabilizing influence can be understood more readilythrough a discussion of certain theoretical bubbles. First, we willconsider the effects of arterial pressure and surface tension on ahypothetical microbubble containing only air.

[0036] Initially, a hypothetical bubble containing only air is prepared.For purposes of discussion, this bubble will initially be considered tohave no LaPlace pressure. Generally, when equilibrated at standardtemperature and pressure (STP), it will have a internal pressure of 760Torr of air and the surrounding fluid air concentration will also beequilibrated at 760 Torr (i.e., the fluid has an air tension of 760Torr). Such a bubble will neither shrink nor grow.

[0037] Next, when the above hypothetical bubble is introduced into thearterial system, the partial pressure of air (or air tension) in theblood (the air pressure at which the blood was saturated with air) willalso be approximately 760 Torr and there will be an arterial pressure(for the purposes of the this discussion at 100 Torr). This totalcreates an external pressure on the bubble of 860 Torr, and causing thegases in the bubble to be compressed until the internal pressureincreases to 860 Torr. There then arises a difference of 100 Torrbetween the air pressure inside the bubble and the air tension of thefluid surrounding the bubble. This pressure differential causes air todiffuse out of the bubble, through its air-permeable surface membrane,causing the bubble to shrink (i.e., lose air) as it strives to reachequilibrium. The bubble shrinks until it disappears.

[0038] Next, consider the additional, and more realistic, effect on thehypothetical bubble of adding the surface tension of the bubble. Thesurface tension of the bubble leads to a LaPlace pressure exerted on gasinside the bubble. The total pressure exerted on the gas inside thebubble is computed through adding the sum of the atmospheric pressure,the arterial pressure and the LaPlace pressure. In a 3 μm bubble asurface tension of 10 dynes per centimeter is attainable with wellchosen surfactants. Thus, the LaPlace pressure exerted on thehypothetical 3 μm bubble is approximately 100 Torr and, in addition, thearterial pressure of 100 Torr is also exerted on the bubble. Therefore,in our hypothetical bubble, the total external pressure applied to thegas inside the bubble is 960 Torr.

[0039] The bubble will be compressed until the pressure of the airinside the bubble rises to 960 Torr. Accordingly, a concentrationdifferential of 200 Torr arises between the air inside the bubble andthe air dissolved in the blood. Therefore, the bubble will rapidlyshrink and disappear even more rapidly than it did in the previous case,as it attempts to reach equilibrium.

[0040] The discovery of the present invention is illustrated byconsidering a third hypothetical microbubble containing air and a gasosmotic agent or a secondary gas. Assume that a theoretical bubble,initially having no arterial pressure and no LaPlace pressure, isprepared having a total pressure of 760 Torr, which is made up of air ata partial pressure of 684 Torr and a perfluorocarbon (“PFC”) as a gasosmotic agent at a partial pressure of 76 Torr. Further, assume that theperfluorocarbon is selected to have one or more traits that make itcapable of acting as an appropriate gas osmotic agent, such as limitedbubble membrane permeability or limited solubility in the externalliquid phase. There is an initial gas osmotic pressure differentialbetween the 684 Torr of air within the bubble and the 760 Torr of airtension outside the bubble (assuming STP) of 76 Torr. This 76 Torrinitial pressure difference is the initial gas osmotic pressure and willcause the bubble to expand. Air from outside of the bubble will diffuseinto and inflate the bubble, driven by the osmotic pressuredifferential, similar to the way water diffuses into a dialysis bagcontaining a starch solution, and inflates the bag.

[0041] The maximum gas osmotic pressure this gas mixture can develop isrelated to the partial pressure of the PFC and the ratio of thepermeability of the PFC to the permeability of the air in thesurrounding fluid. In theory, and as observed experimentally, the bubblewill grow indefinitely as the system attempts to reach osmoticequilibrium between the concentration of air (equivalent to the partialpressure of air) within the bubble and the concentration of airsurrounding the bubble (the air tension).

[0042] When the hypothetical mixed gas bubble is exposed to 100 Torr ofarterial pressure where the blood has a dissolved air tension of 760Torr, the total external pressure will equal 860 Torr (760 Torratmospheric pressure and 100 Torr arterial pressure). The bubble willcompress under the arterial pressure, causing the internal pressure ofthe bubble to reach 860 Torr. The partial pressure of the air willincrease to 774 Torr and the partial pressure of the PFC (the secondgas) will increase to 86 Torr. The air will diffuse out of the bubbleuntil it reaches osmotic equilibrium with the air dissolved in the blood(i.e., 760 Torr) and the partial pressure of the PFC will increase to100 Torr. The partial pressure of the PFC will act to counterbalance thepressure exerted due to the arterial pressure, halting shrinkage of thebubble, in each case, assuming that the permeability of the bubble tothe PFC is negligible.

[0043] When the surface tension or LaPlace pressure component of 100Torr is added (as discussed above with the air bubble), a total of 200Torr additional pressure is exerted on the gas in the bubble. Again, thebubble will compress until and the pressure inside the bubble increasesto 960 Torr (partial pressure of air 864 and partial pressure of PFC96). The air will diffuse from the bubble until it reaches 760 Torr (inequilibrium with the concentration of air the dissolved in the blood)and the partial pressure of the PFC will increase to 200 Torr, where,again, the gas osmotic pressure induced by the PFC will act tocounterbalance the pressure exerted by the LaPlace pressure and thearterial pressure, again, assuming that the membrane permeability of thebubble to the PFC is negligible.

[0044] Similarly, if the partial pressure of air in the bubble is lowerthan the air tension in the surrounding liquid, the bubble will actuallygrow until the PFC is sufficiently diluted by incoming air so that thepressure of air inside and the air tension outside of the bubble areidentical.

[0045] Thus, it can be seen has been shown that bubbles can beeffectively stabilized through the use of combinations of gases, sincethe correct combination of gases will result in a gas osmotic pressuredifferential that can be harnessed to counterbalance the effects of theLaPlace pressure and the arterial pressure exerted on the a gas withinthe bubble in circulating blood.

[0046] Examples of particular uses of the microbubbles of the presentinvention include perfusion imaging of the venous drainage system of theheart, the myocardial tissue, and determination of perfusioncharacteristics of the heart and its tissues during stress or exercisetests, or perfusion defects or changes due to myocardial infarction.Similarly, myocardial tissue can be viewed after oral or venousadministration of drugs designed to increase the blood flow to a tissue.Also, visualization of changes in myocardial tissue due to or duringvarious interventions, such as coronary tissue vein grafting, coronaryangioplasty, or use of thrombolytic agents (TPA or streptokinase) canalso be enhanced. As these contrast agents can be administeredconveniently via a peripheral vein to enhance the visualization of theentire circulatory system, they will also aid in the diagnosis of DeepVein Thrombosis and in the ability to ultrasonically monitor the fetusand the umbilical cord.

[0047] It should, however, be emphasized that these principles haveapplication beyond ultrasound imaging. Indeed, the present invention issufficiently broad to encompass the use of gas osmotic pressure tostabilize bubbles for uses in any systems, including nonbiologicalapplications.

[0048] In a preferred embodiment, the microbubbles of the presentinvention have a surfactant-based bubble membrane. However, theprinciples of the invention can be applied to stabilize microbubbles ofvirtually any type. Thus, mixed gases or vapors of the type describedabove can stabilize albumin based bubbles, polysaccharide basedmicrobubbles, spray dried microsphere derived microbubbles, and thelike. This result is achieved through the entrapment, within the chosenmicrobubble, of a combination of gases, preferably a primary modifiergas or mixture of gases that will dilute a gas osmotic agent to apartial pressure less than the gas osmotic agent's vapor pressure untilthe modifier gas will exchange with gases normally present in theexternal medium. The gas osmotic agent or agents are generallyrelatively hydrophobic and relatively bubble membrane impermeable andalso further possess the ability to develop gas osmotic pressuresgreater than 50, 75, or 100 Torr. In one preferred embodiment, the gasvapor pressure of the gas osmotic agent is preferably less than about760 Torr at 37° C., preferably less than about 750, 740, 730, 720, 710,or 700 Torr, and in some embodiments less than about 650, 600, 500, or400 Torr.

[0049] In preferred embodiments, the vapor pressure of the primarymodifier gas is at least 660 Torr at 37° C. and the vapor pressure ofthe gas osmotic agent is at least 100 Torr at 37° C. For in vivo imagingmean bubble diameters between 1 and 10 μm are preferred, with 3 to 5 μmmost preferred. The invention may in one embodiment also be described asa mixture of a first gas or gases and a second gas or gases withingenerally spherical membranes to form microbubbles, where the first gasand the second gas are respectively present in a molar ratio of about1:100, 1:75, 1:50, 1:30, 1:20, or 1:10 to about 1000:1, 500:1, 250:1,100:1, 75:1 or 50:1, and where the first gas has a vapor pressure of atleast about (760−x) mm Hg at 37° C., where x is the vapor pressure ofthe second gas at 37° C., and where the vapor pressure of each of thefirst and second gases is greater than about 75 or 100 mm Hg at 37° C.

[0050] Microbubbles prepared in accordance with one preferred embodimentof the invention may also possess an additional advantageous property.In one such embodiment, mixtures of nonosmotic gases with osmoticstabilizing gases (or gas osmotic agents) are used to stabilize theresultant bubble size distribution during and immediately afterproduction. Upon generation of the bubbles, the higher LaPlace pressurein smaller bubbles causes diffusion through the liquid phase to thelower LaPlace pressure larger bubbles. This causes the mean sizedistribution to increase above the capillary dimension limit of 5microns over time. This is called disproportionation. When a mixture ofa nonosmotic gas (e.g., air) is used with an osmotic vapor (e.g., C₆F₁₄) a slight reduction in volume of the smaller bubbles, due to airleaving the bubble, concentrates the osmotic gas and increases itsosmotic pressure thus retarding further shrinkage while the largerbubbles increase in volume slightly, diluting the osmotic gas andretarding further growth.

[0051] An additional advantage of using a mixture of an extremely bloodsoluble gases (e.g., 87.5% by volume CO₂) and an osmotic gas mixture(e.g., 28% C₆ F₁₄ vapor+72% air) is that, when injected, these bubblesrapidly shrink due to the loss of CO₂ to the blood. The bubbles, uponinjection, will experience an 87.5% volume decrease due to loss of CO₂.This loss of CO₂ corresponds to a halving of the bubble diameter.Accordingly, one can prepare larger diameter bubbles (e.g., 9 μm), usingsimplified mechanical means, that will shrink to below 5 microns uponinjection. In general, such bubbles will initially be prepared where thefirst gas is present in a ratio of at least 1:1 with respect to thesecond gas, preferably at least 3:2, 2:1, 3:1, 4:1, 5:1, or 10:1. Wherethe microbubble membrane is more permeable to the first gas than to thesecond gas (e.g., the membrane has respective permeabilities to thegases in a ratio of at least about 2:1, 3:1, 4:1, 5:1, or 10:1,preferably even higher, e.g., 20:1, 40:1, or 100:1), the bubblesadvantageously shrink from their original first diameter to an averagesecond diameter of 75% or less of their original diameter quite rapidly(e.g., within one, two, four, or five minutes). Then, when at least onerelatively membrane-permeable gas is present in the aqueous mediumsurrounding the microbubble, the bubble is preferably stabilized at orabout the second diameter for at least about 1 minute, preferably for 2,3, 4, or 5 minutes. In one preferred embodiment, the bubbles maintain asize between about 5 or 6 μm and 1 μm for at least 1, 2, 3, 4, or 5minutes, stabilized by a gas osmotic pressure differential. The gastension in the external liquid is preferably at least about 700 mm Hg.Moreover, a relatively membrane impermeable gas is also in themicrobubble to create such an osmotic pressure differential.

[0052] I. Microbubble Construction

[0053] A. The Aqueous or Other Liquid Phase

[0054] The external, continuous liquid phase in which the bubble residestypically includes a surfactant or foaming agent. Surfactants suitablefor use in the present invention include any compound or compositionthat aids in the formation and maintenance of the bubble membrane byforming a layer at the interface between the phases. The foaming agentor surfactant may comprise a single compound or any combination ofcompounds, such as in the case of co-surfactants.

[0055] Examples of suitable surfactants or foaming agents include: blockcopolymers of polyoxypropylene polyoxyethylene, sugar esters, fattyalcohols, aliphatic amine oxides, hyaluronic acid aliphatic esters,hyaluronic acid aliphatic ester salts, dodecyl poly(ethyleneoxy)ethanol,nonylphenoxy poly(ethyleneoxy)ethanol, hydroxy ethyl starch, hydroxyethyl starch fatty acid esters, dextrans, dextran fatty acid esters,sorbitol, sorbitol fatty acid esters, gelatin, serum albumins, andcombinations thereof.

[0056] In the present invention, preferred surfactants or foaming agentsare selected from the group consisting of phospholipids, nonionicsurfactants, neutral or anionic surfactants, fluorinated surfactants,which can be neutral or anionic, and combinations of such emulsifying orfoaming agents.

[0057] The nonionic surfactants suitable for use in the presentinvention include polyoxyethylene-polyoxypropylene copolymers. Anexample of such class of compounds is Pluronic, such as Pluronic F-68.Also contemplated are polyoxyethylene fatty acids esters, such aspolyoxyethylene stearates, polyoxyethylene fatty alcohol ethers,polyoxyethylated sorbitan fatty acid esters, glycerol polyethyleneglycol oxystearate, glycerol polyethylene glycol ricinoleate,ethoxylated soybean sterols, ethoxylated castor oils, and thehydrogenated derivatives thereof, and cholesterol. Anionic surfactants,particularly fatty acids (or their salts) having 12 to 24 carbon atoms,may also be used. One example of a suitable anionic surfactant is oleicacid, or its salt, sodium oleate.

[0058] It will be appreciated that a wide range of surfactants can beused. Indeed, virtually any surfactant or foaming agent (including thosestill to be developed) capable of facilitating formation of themicrobubbles can be used in the present invention. The optimumsurfactant or foaming agent or combination thereof for a givenapplication can be determined through empirical studies that do notrequire undue experimentation. Consequently, one practicing the art ofthe present invention should choose the surfactant or foaming agents orcombination thereof based upon such properties as biocompatibility ortheir non-Newtonian behavior.

[0059] The blood persistence of a contrast agent is inverselyproportional to the LaPlace pressure which is proportional to thesurface tension of the bubble. Reduced surface tension, therefore,increases blood persistence. Surfactants that form ordered structures(multilaminar sheets and rods) in solution and produce non-Newtonianviscoelastic surface tensions are especially useful. Such surfactantsinclude many of the sugar based surfactants and protein or glycoproteinsurfactants (including bovine, human, or other lung surfactants). Onepreferred type of such surfactant has a sugar or other carbohydrate headgroup, and a hydrocarbon or fluorocarbon tail group. A large number ofsugars are known that can function as head groups, including glucose,sucrose, mannose, lactose, fructose, dextrose, aldose, and the like. Thetail group preferably has from about 2 or 4 to 20 or 24 carbon atoms,and may be, for example, a fatty acid group (branched or unbranched,saturated or unsaturated) covalently bound to the sugar through an esterbond. The surface tension of bubbles produced with these surfactantsgreatly decreases as the surface is compressed by shrinkage of thebubble (e.g., when the bubble shrinks), and it is increased as thesurface area of the bubble is increased (e.g., when the bubble grows).This effect retards disproportionation, which leads to narrower sizedistribution and longer persisting bubbles in the vial and in vivo. Apreferred surfactant mixture that has the properties associated withnon-Newtonian viscoelasticity includes a nonionic surfactant or otherfoaming surfactant in combination with one of the non-Newtonianviscoelastic surfactant such as one of the sugar esters (e.g. 2%Pluronic F-68 plus 1% sucrose stearate). Often the ratio of the nonionicsurfactant to the non-Newtonian surfactant is from about 5:1 to about1:5, with the surfactants together (whether non-Newtonian or moreconventional) comprising 0.5 to 8%, more preferably about 1 to 5% (w/v)of the microbubble-forming liquid mixture.

[0060] The lowering of surface tension in small bubbles, counter totypical LaPlace pressure, allows the use of more efficient gas osmoticagents such as higher molecular weight perfluorocarbons as the gasosmotic agent. With conventional surfactants, the higher molecularweight PFCs will condense at the high bubble pressures. Without theseefficient surfactants higher boiling less membrane permeable PFCs, e.g.C₆ F₁₄, would be extremely difficult.

[0061] One may also incorporate other agents within the aqueous phase.Such agents may advantageously include conventional viscosity modifiers,buffers such as phosphate buffers or other conventional biocompatiblebuffers or pH adjusting agents such as acids or bases, osmotic agents(to provide isotonicity, hyperosmolarity, or hyposmolarity). Preferredsolutions have a pH of about 7 and are isotonic. However, when CO₂ isused as a first gas in a bubble designed to shrink rapidly to a firstsize, a basic pH can facilitate rapid shrinkage by removing CO₂ as itleaves the bubble, preventing a buildup of dissolved CO₂ in the aqueousphase.

[0062] B. The Gas Phase

[0063] A major aspect of the present invention is in the selection ofthe gas phase. As was discussed above, the invention relies on the useof combinations of gases to harness or cause differentials in partialpressures and to generate gas osmotic pressures, which stabilize thebubbles. The primary modifier gas is preferably air or a gas present inair. Air and/or fractions thereof are also present in normal blood andserum. Where the microbubbles are to be used in an environment differentfrom blood, the primary modifier gas is preferably selected from gasesnormally present in the external medium. Another criteria is the easewith which the primary modifier gas is diffused into or out of thebubbles. Typically, air and/or fractions thereof are also readilypermeable through conventional flexible or rigid bubble surfaces. Thesecriteria, in combination, allow for the rapid diffusion of the primarymodifier gas into or out of the bubbles, as required.

[0064] Modifier gases not present in the external medium can also beused. However, in this case the bubble will initially grow or shrink(depending on the relative permeability and concentrations of theexternal gases to the modifier) as the external gases replace theoriginal modifier gas. If, during this process, the gas osmotic agenthas not condensed, the bubble will remain stable.

[0065] The gas osmotic agent is preferably a gas that is less permeablethrough the bubble's surface than the modifier. It is also preferablethat the gas osmotic agent is less soluble in blood and serum.Therefore, it will now be understood that the gas osmotic agent can be agas at room or body temperature or it can ordinarily be a liquid at bodytemperature, so long as it has a sufficient partial or vapor pressure atthe temperature of use to provide the desired osmotic effect.

[0066] Accordingly, fluorocarbons or other compounds that are not gasesat room or body temperature can be used, provided that they havesufficient vapor pressure, preferably at least about 50 or 100 Torr atbody temperature, or more preferably at least about 150 or 200 Torr. Itshould be noted that where the gas osmotic agent is a mixture of gases,the relevant measure of vapor pressure is the vapor pressure of themixture, not necessarily the vapor pressure of the individual componentsof the mixed gas osmotic agent.

[0067] It is also important that where a perfluorocarbon is used as theosmotic agent within a bubble, the particular perfluorocarbon does notcondense at the partial pressure present in the bubble and at bodytemperature. Depending on the relative concentrations of the primarymodifier gas and the gas osmotic agent, the primary modifier gas mayrapidly leave the bubble causing it to shrink and concentrate thesecondary gas osmotic agent. Such shrinking may occur until the gasosmotic pressure equals the external pressure on the bubble (maximumabsolute arterial pressure) plus the LaPlace pressure of the bubbleminus the air tension, or air saturation tension, of the blood(essentially one atmosphere). Thus the condensing partial pressure ofthe resulting gas mixture at 37° C. must be above the equilibriumpartial pressure, discussed above, of the osmotic agent.

[0068] Representative fluorocarbons meeting these criteria and inincreasing ability to stabilize microbubbles are as follows:

CCl₂F₂<CF₄, CHClF₂<C₄F₁₀, N(C₂F₅)₃<C₅F₁₂<C.₆F₁₄

[0069] Accordingly, it will be understood that PFC's with eight carbonsatoms or fewer (37° C. vapor pressures greater than 80 mm Hg) arepreferred. As will also be understood, however, it is possible toconstruct larger molecules with increased volatility through theaddition of heteroatoms and the like. Therefore, the determination ofthe optimal secondary gas osmotic agent or gases agents is not sizelimited, but, rather, is based upon its ability to retain its vaporphase at body temperature and while providing a gas osmotic pressureequal to at least the sum of the arterial and LaPlace pressures.

[0070] A listing of some compounds possessing suitable solubility andvapor pressure criteria is provided in Table I: TABLE I perfluoropropanes, C₃F₈ perfluoro butanes, C₄F_(.10) perfluoro cyclo butanes,C₄F_(.8) perfluoro pentanes, C₅F₁₂ perfluoro cyclo pentanes, C_(.5)F₁₀perfluoro methylcyclobutanes, C₅F₁₀ perfluoro hexanes, C₆F_(.4)perfluoro cyclohexanes, C₆F₁₂ perfluoro methyl cyclopentanes, C₆F₁₂perfluoro dimethyl cyclobutanes, C₆F₁₂ perfluoro heptanes, C₇F_(.16)perfluoro cycloheptanes, C₇F₁₄ perfluoro methyl cyclohexanes, C₇F₁₄perfluoro dimethyl cyclopentanes, C₇F₁₄ perfluoro trimethylcyclobutanes, C₇F₁₄ perfluoro triethylamines, N(C₂F₅)₃

[0071] It will be appreciated that one of ordinary skill in the art canreadily determine other compounds that would perform suitably in thepresent invention that do not meet both the solubility and vaporpressure criteria, described above. Rather, it will be understood thatcertain compounds can be considered outside the preferred range ineither solubility or vapor pressure, if such compounds compensate forthe aberration in the other category and provide a superior insolubilityor low vapor pressure.

[0072] It should also be noted that for medical uses the gases, both themodifier gas and the gas osmotic agent, should be biocompatible or notbe physiologically deleterious. Ultimately, the microbubbles containingthe gas phase will decay and the gas phase will be released into theblood either as a dissolved gas or as submicron droplets of thecondensed liquid. It will be understood that gases will primarily beremoved from the body through lung respiration or through a combinationof respiration and other metabolic pathways in the reticuloendothelialsystem.

[0073] Appropriate gas combinations of the primary modifier andsecondary gases can be ascertained empirically without undueexperimentation. Such empirical determinations are described in theExamples.

[0074] When an efficient surfactant, e.g., bovine lung surfactant, isemployed to produce a large diameter bubble with a low surface tension,the LaPlace pressure is very low. When perfluorooctylbromide (PFOB)saturated air is inside the bubble and the bubble is exposed to air or aliquid nearly saturated with air (e.g., equilibrated with air) the gasosmotic pressure is greater than the LaPlace pressure and therefore thebubble grows. With smaller diameter bubbles the LaPlace pressure ishigher and therefore the bubble shrinks and collapses. This shrinkage isat a reduced rate being driven by the difference between the LaPlacepressure minus reduced by the gas osmotic pressure. When small diameterbubbles are created by sonicating gas or gas vapor mixtures in a lowsurface tension surfactant solution, e.g., 2% pluronic F-68 plus 1%sucrose stearate, the time the bubbles persist in vitro, as observed bymicroscope, and in vivo as observed by Doppler ultrasound imaging of arabbit's kidney post intravenous injection, correlated with the abovegas osmotic pressure comparison.

[0075] In the rabbit kidney Doppler experiment (Example III), contrastenhancement was observed for up to 10 minutes with perfluorohexane/airmixtures in the bubbles compared with the instantaneous disappearance ofcontrast with pure air microbubbles. Thus, these perfluorochemicals arecapable of exerting gas osmotic pressures that nearly counterbalance theLaPlace pressure and create functional ultrasound microbubble contrastagents.

[0076] A surprising discovery was that mixtures of PFCs, e.g., C₄ F₁₀(as a combination modifier gas and a gas osmotic agent) saturated withC₆ F₁₄ vapor (as the main gas osmotic agent), can stabilize the bubblefor longer times than either component alone. This is because C₄ F₁₀ isa gas at body temperature (and, thus, can act as both a modifier gas anda gas osmotic agent) has a somewhat reduced membrane permeability and itis only slightly soluble in C₆ F₁₄ at body temperature. In thissituation the gas osmotic pressures of both agents are added together,leading to increased bubble persistence over that of air/C₆ F₁₄ onlymixtures. It is possible that the condensing point of the longerpersisting higher molecular weight C₆ F₁₄ component is increased,allowing a larger maximum gas osmotic pressure to be exerted. Othermixtures of PFCs will perform similarly. Preferred mixtures of PFCs willhave ratios of 1:10 to 10:1, and include such mixtures asperfluorobutane/perfluorohexane and perfluorobutane/perfluoropentane.These preferred fluorochemicals can be branched or straight chain.

[0077] As was discussed above, we have also discovered that mixtures ofnonosmotic gases in combination with the gas osmotic agent act tostabilize the size distribution of the bubbles before and afterinjection. Upon generation of the bubbles, the higher LaPlace pressuresin smaller bubbles causes diffusion through the liquid phase to thelower LaPlace pressure larger bubbles. This causes the mean sizedistribution to increase above the capillary dimension limit of 5microns with time. This is called disproportionation.

[0078] However, when a mixture of a modifier gases (e.g., air or carbondioxide) are used with a gas osmotic agent (e.g., C₆ F₁₄) a slightreduction in volume of the smaller bubbles, due to one of the modifiergases leaving the bubble, will concentrate the osmotic gas and increasesits osmotic pressure, thus, retarding further shrinkage. On the otherhand, the larger bubbles will increase in volume slightly, diluting theosmotic gas and also retarding further growth.

[0079] An additional advantage of using a mixture of an extremely bloodsoluble gas (e.g., 75% through 87.5% by volume CO₂) and an osmotic gasmixture (e.g. 28% C₆ F₁₄ vapor and 72% air) is that when injected, thesebubbles rapidly shrink due to the loss of CO₂ to the blood. Carbondioxide leaves particularly fast due to a specific plasma enzyme thatcatalyzes its dissolution. An 87.5% volume decrease due to loss of CO₂corresponds with a halving of the bubble diameter. Accordingly, largercan be produced which will shrink to an appropriate size (i.e., 5microns) upon injection or exposure to a solution with a basic oralkaline pH.

[0080] Accordingly, we have discovered that through use of a gas that isrelatively hydrophobic and that has a relatively low membranepermeability, the rate of contrast particle decay can be reduced. Thus,through reducing the particle decay rate, the microbubbles' half livesare increased and contrast enhancement potential is extended.

[0081] II. Other Components.

[0082] It will be understood that other components can be included inthe microbubble formulations of the present invention. For example,osmotic agents, stabilizers, chelators, buffers, viscosity modulators,air solubility modifiers, salts, and sugars can be added to fine tunethe microbubble suspensions for maximum life and contrast enhancementeffectiveness. Such considerations as sterility, isotonicity, andbiocompatibility may govern the use of such conventional additives toinjectable compositions. The use of such agents will be understood tothose of ordinary skill in the art and the specific quantities, ratios,and types of agents can be determined empirically without undueexperimentation.

[0083] III. Formation of the Microbubbles of the Present Invention.

[0084] There are a variety of methods to prepare microbubbles inaccordance with the present invention. Sonication is preferred for theformation of microbubbles, i.e., through an ultrasound transmittingseptum or by penetrating a septum with an ultrasound probe including anultrasonically vibrating hypodermic needle. However, it will beappreciated that a variety of other techniques exist for bubbleformation. For example, gas injection techniques can be used, such asventuri gas injection.

[0085] Other methods for forming microbubbles include formation ofparticulate microspheres through the ultrasonication of albumin or otherprotein as described in European Patent Application 0,359,246 byMolecular Biosystems, Inc.; the use of tensides and viscosity increasingagents as described in U.S. Pat. No. 4,446,442; lipid coated,non-liposomal, microbubbles as is described in U.S. Pat. No. 4,684,479;liposomes having entrapped gases as is described in U.S. Pat. Nos.5,088,499 and 5,123,414; and the use of denatured albumin particulatemicrospheres as is described in U.S. Pat. No. 4,718,433. The disclosureof each of the foregoing patents and applications is hereby incorporatedby reference.

[0086] Any of the above methods can be employed with similar success toentrain the modifier gases and gas osmotic agents of the presentinvention. Moreover, it is expected that similar enhancement inlongevity of the bubbles created will be observed through use of theinvention.

[0087] Sonication can be accomplished in a number of ways. For example,a vial containing a surfactant solution and gas in the headspace of thevial can be sonicated through a thin membrane. Preferably, the membraneis less than about 0.5 or 0.4 mm thick, and more preferably less thanabout 0.3 or even 0.2 mm thick, i.e., thinner than the wavelength ofultrasound in the material, in order to provide acceptable transmissionand minimize membrane heating. The membrane can be made of materialssuch as rubber, Teflon, mylar, urethane, aluminized film, or any othersonically transparent synthetic or natural polymer film or film formingmaterial. The sonication can be done by contacting or even depressingthe membrane with an ultrasonic probe or with a focused ultrasound“beam.” The ultrasonic probe can be disposable. In either event, theprobe can be placed against or inserted through the membrane and intothe liquid. Once the sonication is accomplished, the microbubblesolution can be withdrawn from and vial and delivered to the patient.

[0088] Sonication can also be done within a syringe with a low powerultrasonically vibrated aspirating assembly on the syringe, similar toan inkjet printer. Also, a syringe or vial may be placed in andsonicated within a low power ultrasonic bath that focuses its energy ata point within the container.

[0089] Mechanical formation of microbubbles is also contemplated. Forexample, bubbles can be formed with a mechanical high shear valve (ordouble syringe needle) and two syringes, or an aspirator assembly on asyringe. Even simple shaking may be used. The shrinking bubbletechniques described herein are particularly suitable for mechanicallyformed bubbles, having lower energy input than sonicated bubbles. Suchbubbles will typically have a diameter much larger than the ultimatelydesired biocompatible imaging agent, but can be made to shrink to anappropriate size in accordance with the present invention.

[0090] In another method, microbubbles can be formed through the use ofa liquid osmotic agent emulsion supersaturated with a modifier gas atelevated pressure introduced into in a surfactant solution. Thisproduction method works similarly to the opening of soda pop, where thegas foams upon release of pressure forming the bubbles.

[0091] In another method, bubbles can be formed similar to the foamingof shaving cream, where perfluorobutane, freon, or another like materialthat boils when pressure is released. However, in this method it isimperative that the emulsified liquid boils sufficiently low or that itcontain numerous bubble nucleation sites so as to prevent superheatingand supersaturation of the aqueous phase. This supersaturation will leadto the generation of a small number of large bubbles on a limited numberof nucleation sites rather than the desired large number of smallbubbles (one for each droplet).

[0092] In still another method, dry void-containing particles or otherstructures (such as hollow spheres or honeycombs) that rapidly dissolveor hydrate, preferably in an aqueous solution, e.g., albumin, microfinesugar crystals, hollow spray dried sugar, salts, hollow surfactantspheres, dried porous polymer spheres, dried porous hyaluronic acid, orsubstituted hyaluronic acid spheres, or even commercially availabledried lactose microspheres can be stabilized with a gas osmotic agent.

[0093] For example, a spray dried surfactant solution can be formulatedto obtain 5 micron or larger hollow spheres and packaged in a vialfilled with an osmotic gas or a desired gas mixture as described herein.The gas will diffuse into the spheres. Diffusion can be aided bypressure or vacuum cycling. When reconstituted with a sterile solutionthe spheres will rapidly dissolve and leave osmotic gas stabilizedbubbles in the vial. In the alternative, a lyophilized cake ofsurfactant and bulking reagents produced with a fine pore structure canbe placed in a vial with a sterile solution and a head spaced with anosmotic gas mixture. The solution can be frozen rapidly to produce afine ice crystal structure and, therefore, upon lyophilization producesfine pores (voids where the ice crystals were removed).

[0094] Alternatively, any dissolvable or soluble void-forming structuresmay be used. In this embodiment, where the void-forming material is notmade from or does not contain surfactant, both surfactant and liquid aresupplied into the container with the structures and the desired gas orgases. Upon reconstitution these voids trap the osmotic gas and, withthe dissolution of the solid cake, form microbubbles with the gas orgases in them.

[0095] It will be appreciated that kits can be prepared for use inmaking the microbubble preparations of the present invention. These kitscan include a container enclosing the gas or gases described above forforming the microbubbles, the liquid, and the surfactant. Alternatively,the container can contain the void forming material and the gas orgases, and the surfactant and liquid can be added to form themicrobubbles. Alternatively, the surfactant can be present with theother materials in the container, and only the liquid needs to be addedin order to form the microbubbles. Where a material necessary forforming the microbubbles is not already present in the container, it canbe packaged with the other components of the kit, preferably in a formor container adapted to facilitate ready combination with the othercomponents of the kit.

[0096] The container used in the kit may be of the type describedelsewhere herein. In one embodiment, the container is a conventionalseptum-sealed vial. In another, it has a means for directing orpermitting application of sufficient bubble forming energy into thecontents of the container. This means can comprise, for example, thethin web or sheet described previously.

[0097] Any of the microbubble preparations of the present invention maybe administered to a vertebrate, such as a bird or a mammal, as acontrast agent for ultrasonically imaging portions of the vertebrate.Preferably, the vertebrate is a human, and the portion that is imaged isthe vasculature of the vertebrate. In this embodiment, a small quantityof microbubbles (e.g., 0.1 ml/Kg based on the body weight of thevertebrate) is introduced intravascularly into the animal. Otherquantities of microbubbles, such as from about 0.005 ml/Kg to about 1.0ml/Kg, can also be used. Imaging of the heart, arteries, veins, andorgans rich in blood, such as liver, lungs, and kidneys can beultrasonically imaged with this technique.

[0098] The foregoing description will be more fully understood withreference to the following Examples. Such Examples, are, however,exemplary of preferred methods of practicing the present invention andare not limiting of the scope of the invention or the claims appendedhereto.

EXAMPLE I Preparation of Microbubbles Through Sonication

[0099] Microbubbles with an average number weighted size of 5 micronswere prepared by sonication of an isotonic aqueous phase containing 2%Pluronic F-68 and 1% sucrose stearate as surfactants, air as a modifiergas and perfluorohexane as the gas osmotic agent.

[0100] In this experiment, 1.3 ml of a sterile water solution containing0.9% NaCl, 2% Pluronic F-68 and 1% sucrose stearate was added to a 2.0ml vial. The vial had a remaining head space of 0.7 ml initiallycontaining air. Air saturated with perfluorohexane vapor (220 torr ofperfluorohexane with 540 torr of air) at 25 degrees C. was used to flushthe headspace of the vial. The vial was sealed with a thin 0.22 mmpolytetrafluoroethylene (PTFE) septum. The vial was turned horizontally,and a ⅛″ (3 mm) sonication probe attached to a 50 watt sonicator modelVC₅₀, available from Sonics & Materials was pressed gently against theseptum. In this position, the septum separates the probe from thesolution. Power was then applied to the probe and the solution wassonicated for 15 seconds, forming a white solution of finely dividedmicrobubbles, having an average number weighted size of 5 microns asmeasured by Horiba LA-700 laser light scattering particle analyzer.

EXAMPLE II Measurement of In-Vitro Size of Microbubbles

[0101] The in-vitro size of the microbubbles prepared in Example I wasmeasured by laser light scattering. Studies of bubbles were conductedwhere the microbubbles were diluted into a 4% dextrose water solution(1:50) circulating through a Horiba LA-700 laser light scatteringanalyzer. The average microbubbles size was 5 microns and doubled insize in 25 minutes.

[0102] Interestingly, microbubbles prepared through the same method inExample I without the use of a gas osmotic agent (substituting air forthe perfluorohexane/air mixture) had an average size of 11 microns andgave only background readings on the particle analyzer at 10 seconds.

EXAMPLE III Measurement of In-Vivo Lifetime of Microbubbles

[0103] The lifetimes of microbubbles prepared in accordance with ExampleI were measured in rabbits through injecting 0.2 ml of freshly formedmicrobubbles into the marginal ear vein of a rabbit that was underobservation with a Accuson 128XP/5 ultrasound imaging instrument with a5 megahertz transducer. Several tests were conducted, during whichimages of the heart, inferior vena cava/aorta, and kidney were obtainedwhile measuring the time and extent of the observable contrastenhancement. The results are presented in the following Table II: TABLEII TIME TO MINIMUM TIME MAX. USABLE TIME TO NO ORGAN DOSE INTENSITYINTENSITY ENHANCEMENT Heart 0.1 ml/Kg 7-10 sec. 8-10 min. 25 minIVC/Aorta 0.1 ml/Kg 7-10 sec. 8-10 min. 25 min Kidney 0.1 ml/Kg 7-10sec. 8-10 min 25 min

[0104] In Table III, a comparison of microbubbles prepared in anidentical fashion without the use of an osmotic gas is presented (onlyair was used). Note that sporadic reflections were observed only in theright heart ventricle during the injection but disappeared immediatelypost dosing. TABLE III TIME TO TIME TO MINIMUM MAXIMUM USABLE TIME TO NOORGAN DOSE INTENSITY INTENSITY ENHANCEMENT Heart 0.1 ml/Kg 0 0 0IVC/Aorta 0.1 ml/Kg 0 0 0 Kidney 0.1 ml/Kg 0 0 0

[0105] The use of an osmotic or gas osmotic agent dramatically increasedthe length of time for which microbubbles are visible.

EXAMPLE IV Preparation of Mixed Osmotically Stabilized Microbubbles

[0106] Microbubbles with an average number weighted size of 5 micronswere prepared by sonication of an isotonic aqueous phase containing 2%Pluronic F-68 and 1% sucrose stearate as surfactants and mixtures ofperfluorohexane and perfluorobutane as the gas osmotic agents.

[0107] In this experiment, 1.3 ml of a sterile water solution containing0.9% NaCl and 2% Pluronic F-68 was added to a 2.0 ml vial. The vial hada remaining head space of 0.7 ml, initially containing air. An osmoticgas mixture of perfluorohexane, 540 Torr and perfluorobutane at 220 Torrwas used to flush the headspace before sealing with a thin 0.22 mm PTFEseptum. The vial was sonicated as in Example I, forming a white solutionof finely divided microbubbles, having an average particle size of 5microns as measured by a Horiba LA-700 laser light scattering particleanalyzer. This procedure was repeated twice more, once with pureperfluorobutane and then with a 540 Torr air+220 Torr perfluorohexanemixture. Vascular persistence of all three preparations was determinedby ultrasound imaging of a rabbit post I.V. injection and are listedbelow: 1.5 minutes perfluorobutane 2 minutes perfluorohexane + air 3minutes perfluorbutane + perfluorohexane

[0108] The mixture of perfluorocarbons persisted longer than eitheragent alone.

EXAMPLE V Preparation of Gas Osmotically Stabilized Microbubbles fromSoluble Spray Dried Spheres

[0109] Gas osmotically stabilized microbubbles were prepared bydissolving hollow spray dried lactose spheres, filled with an airperfluorohexane vapor mixture, in a surfactant solution.

[0110] Spray dried spheres of lactose with a mean diameter ofapproximately 100 micron and containing numerous 10 to 50 microncavities, was obtained from DMV International under the trade name ofPharmatose DCL-11. Ninety milligrams of the lactose spheres was placedin a 2.0 ml vial. The porous spheres were filled with a mixture of 220Torr perfluorohexane and 540 Torr air by cycling the gas pressure in thevial between one atmosphere and ½ atmosphere a total of 12 times over 5minutes. A surfactant solution containing 0.9% sodium chloride, 2%Pluronic-F₆₈ and 1% sucrose stearate was warmed to approximately 45° C.,to speed the dissolution of the lactose, before injecting 1.5 ml of thewarmed solution into the vial. The vial was then gently agitated byinversion for approximately 30 seconds to dissolve the lactose beforeinjecting the microbubbles thus prepared into the Horiba LA-700 particleanalyzer. A 7.7 micron volume weighted median diameter was measuredapproximately one minute after dissolution. The diameter of thesemicrobubbles remained nearly constant, changing to a median diameter of7.1 microns in 10 minutes. When the experiment was repeated with airfilled lactose, the particle analyzer gave only background readings oneminute after dissolution, thus demonstrating that gas osmoticallystabilized microbubbles can be produced by the dissolution of gas-filledcavity-containing structures.

EXAMPLE VI Preparation of Larger Bubbles that Shrink to a Desired Size

[0111] Microbubbles with an average volume weighted size of 20 micronsshrinking to 2 microns were prepared by sonication of an isotonicaqueous phase containing 2% Pluronic F-68 as the surfactant, CO₂ as adiluent gas and perfluorohexane as the gas osmotic agent.

[0112] In this experiment, 1.3 ml of a sterile water solution containing0.9% NaCl, 2% Pluronic F-68 and 1% sucrose stearate was added to a 2.0ml vial. The vial had a remaining head space of 0.7 ml initiallycontaining air. A mixture of air saturated with perfluorohexane at 25degrees C. diluted by a factor of 10 with CO₂ (684 Torr CO₂+54 Torrair+22 Torr perfluorohexane) was used to flush the head space. The vialwas sealed with a thin 0.22 mm PTFE septum. The vial was sonicated as inExample I, forming a white solution of finely divided microbubbles,having an average particle size of 28 microns as measured by HoribaLA-700 laser light scattering analyzer. In the 4% dextrose+0.25 mM NaOHsolution of the Horiba, the average bubble diameter rapidly shrank in 2to 4 minutes from 28 microns to 5 to 7 microns, and then remainedrelatively constant, reaching 2.6 micron after 27 minutes. This isbecause the CO₂ leaves the microbubbles by dissolving into the waterphase.

EXAMPLE VII Perfluoroheptane Stabilized Microbubble In Vitro Experiment

[0113] Microbubbles were prepared as in Example I above employingperfluoroheptane saturated air (75 torr plus 685 torr air) and weremeasured as in Example II above. The average number weighted diameter ofthese microbubbles was 7.6 micron, one minute after circulation, and 2.2microns after 8 minutes of circulation. This persistence, compared tothe near immediate disappearance of microbubbles containing only air,demonstrates the gas osmotic stabilization of perfluoroheptane.

EXAMPLE VIII Perfluorotripropyl Amine Stabilized Microbubble In vivoExperiment

[0114] Microbubbles were prepared as in Example I above, employingperfluorotripropyl amine saturated air and were assessed as in ExampleIII above. The usable vascular persistence of these microbubbles wasfound to be 2.5 minutes, thus demonstrating the gas osmoticstabilization of perfluorotripropyl amine.

EXAMPLE IX Effect of a Non Newtonian Viscoelastic Surfactant—SucroseStearate

[0115] Microbubbles were prepared as in Example I above employing 0.9%NaCl, 2% Pluronic F-68 and 2% sucrose stearate as the surfactant andwith perfluoropropane saturated air and perfluorohexane saturated air inthe headspace. These two preparations were repeated with the samesurfactant solution minus sucrose stearate. All four microbubblepreparations were assessed as in Example III above. The usable vascularpersistence of these microbubbles are listed below:

[0116] 2% Pluronic F-68+2% sucrose stearate persistence

[0117] 2 minutes perfluoropropane

[0118] 4 minutes perfluorohexane

[0119] 2% Pluronic F-68 only persistence

[0120] 2 minutes perfluoropropane

[0121] 1 minute perfluorohexane

[0122] As seen above, the reduced surface tension made possible by thenon-Newtonian viscoelastic properties of sucrose stearate prevented theless volatile perfluorohexane from condensing, allowing perfluorohexanemicrobubbles of longer persistence to be produced.

[0123] The foregoing description details certain preferred embodimentsof the present invention and describes the best mode contemplated. Itwill be appreciated, however, that no matter how detailed the foregoingappears in text, the invention can be practiced in many ways and theinvention should be construed in accordance with the appended Claims andany equivalents thereof.

What is claimed is:
 1. A microbubble for the in vivo transport ofphysiological gases wherein the microbubble comprises at least onefluorocarbon gas and at least one modifier gas wherein the microbubblegrows and shrinks to maintain osmotic equilibrium with the physiologicalgas saturation of the surrounding external medium.
 2. The microbubble ofclaim 1 wherein the modifier gas saturation level changes in the bubbleas the microbubble circulates.
 3. The microbubble of claim 1 wherein thesurrounding external medium is blood.
 4. The microbubble of claim 1wherein the microbubble comprises at least one fluorocarbon gas and atleast one modifier gas while in vivo.
 5. The microbubble of claim 1wherein the physiological gases are at least one gas selected from thegroup consisting of oxygen, nitrogen and carbon dioxide.
 6. Themicrobubble of claim 1 wherein the microbubble grows when the modifiergas of the microbubble exchanges with gases present in the surroundingexternal medium.
 7. The microbubble of claim 6 wherein the physiologicalgas present in the surrounding external medium is oxygen.
 8. Themicrobubble of claim 6 wherein the physiological gas present in thesurrounding external medium is air.
 9. The microbubble of claim 1wherein the at least one fluorocarbon gas is selected from the groupconsisting of perfluoropropanes, perfluorobutanes,perfluorocyclobutanes, perfluoropentanes, perfluorocyclopentanes,perfluoromethylcyclopentanes, perfluorohexanes, perfluorocyclohexanes,perfluoromethylcyclopentanes, perfluorodimethylcyclobutanes,perfluoroheptanes, perfluorocycloheptanes, perfluoromethylcyclohexanes,perfluorodimethylcyclopentanes, perfluorotrimethylcyclobutanes,perfluorotriethylamines, and sulfur hexafluoride.
 10. A microbubble forin vivo delivery of physiological gases to an organism or tissues of anorganism wherein the microbubble comprises at least one fluorocarbon gasand at least one modifier gas comprising oxygen wherein the microbubblegrows or shrinks in diameter to maintain osmotic equilibrium with thesurrounding external medium.
 11. The microbubble composition of claim 10wherein the fluorocarbon gas is perfluorohexane.
 12. The microbubble ofclaim 10 wherein the microbubble grows in diameter to maintain osmoticequilibrium of oxygen within the microbubble with the oxygen in thesurrounding medium.
 13. The microbubble of claim 10 wherein thesurrounding external medium is blood.
 14. The microbubble of claim 10wherein the one fluorocarbon gas is selected from the group consistingof perfluoropropanes, perfluorobutanes, perfluorocyclobutanes,perfluoropentanes, perfluorocyclopentanes, perfluoromethylcyclopentanes,perfluorohexanes, perfluorocyclohexanes, perfluoromethylcyclopentanes,perfluorodimethylcyclobutanes, perfluoroheptanes,perfluorocycloheptanes, perfluoromethylcyclohexanes,perfluorodimethylcyclopentanes, perfluorotrimethylcyclobutanes,perfluorotriethylamines, and sulfur hexafluoride.
 15. The microbubble ofclaim 10 wherein the microbubble further comprises a membrane.
 16. Themicrobubble of claim 11 wherein the microbubble further comprises amembrane.
 17. A microbubble composition for the in vivo transport ofphysiological gases wherein the microbubble comprises at least onefluorocarbon gas and at least one modifier gas wherein the microbubblefirst shrinks as a result of loss of the modifier gas to the surroundingmedium and then grows as the microbubble gains osmotic equilibrium withthe physiological gas saturation of the surrounding medium.
 18. Themicrobubble composition of claim 17 wherein the modifier gas is selectedfrom the group consisting of oxygen, nitrogen and carbon dioxide. 19.The microbubble composition of claim 17 wherein the transportedphysiological gas is oxygen.
 20. The microbubble composition of claim 17wherein the fluorocarbon gas is selected from the group consisting ofperfluoropropanes, perfluorobutanes, perfluorocyclobutanes,perfluoropentanes, perfluorocyclopentanes, perfluoromethylcyclopentanes,perfluorohexanes, perfluorocyclohexanes, perfluoromethylcyclopentanes,perfluorodimethylcyclobutanes, perfluoroheptanes,perfluorocycloheptanes, per fluoromethylcyclohex anes,perfluorodimethylcyclopentanes, perfluorotrimethylcyclobutanes,perfluorotriethylamines, and sulfur hexafluoride.